Browsing by Author "Timol, Zaheer"

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    Chemical and conformational studies of bacterial cell surface polysaccharide repeating units
    (2017) Timol, Zaheer; Ravenscroft, Neil Kuttel, Michelle Mary; Gammon, David W
    Abstract Bacterial cell surface polysaccharides are primarily present as lipopolysaccharides or capsular polysaccharides. They are used by cells for both structure and function and have been shown to be a virulence factor of bacterial pathogens. Cell surface polysaccharides are widely utilised as antigenic components in vaccines and play an important role in the protection against numerous diseases including meningococcal disease and shigellosis. This study is composed of two parts: a computational section, which investigates the capsular polysaccharide (CPS) repeating unit (RU) conformations of meningococcal Y and W CPS vaccines and a second experimental component that involves synthetic studies toward the O-specific polysaccharide (O-SP) RU of Shigella sonnei. The CPS RU of MenY [→6)-α-D-Glc(1→4)-α-D-NeuNAc-(2→] and MenW [→6)-α-D-Gal(1→4)-α-D-NeuNAc-(2→] differ only in the orientation of the C-4 hydroxyl: equatorial in MenY and axial in MenW. However, groups Y and W CPS vaccines have different levels of antibody cross-protection. The purpose of the computational study was to determine if these observed differences may be attributed to CPS RU conformation. Potential of mean force calculations were applied to disaccharide RUs of MenY and MenW, and larger three repeating units (3RU) were simulated with molecular dynamics (MD) in solution. The molecular modelling showed that differences in RU conformation between the meningococcal groups arise primarily due to the structural differences between glucose and galactose; affecting the behaviour and orientation of the 2→6 dihedral linkage. The 2→6 linkages in the MenY 3RU adopt a single preferred orientation and consequently it has a single dominant molecular conformation. In contrast, the 2→6 linkages in the MenW 3RU move frequently between different rotameric conformations resulting in multiple conformational families. These results indicate significant conformational differences between the MenY and MenW CPS RUs, which may account for the different levels of cross-protection observed. The synthetic component was part of a larger study to develop a novel route towards the O-SP RU of S. sonnei for use in biological testing and physicochemical characterisation for vaccine development. The O-SP RU of S. sonnei is →4-α-L-AltNAc-(1→3)-β-D-FucNAc4N(1→. The multi-step synthesis was performed using known methodology as well as methods developed by the research group. The key 2,3-oxazolidinone protected intermediate was successfully synthesised in good yields and due to time constraints the final product synthesised was two steps away from the protected FucNAc4N residue. Additional studies were performed on the 2,3-oxazolidinone intermediate as part of a divergent synthesis strategy toward the AltNAcA residue of S. sonnei. Reactions were conducted whereby β and α derivatives of the 2,3-oxazolidinone intermediate were successfully synthesised in large scale and good yields for further studies to be performed by the group. i
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    Modelling and validation of bacterial O-antigen conformations: ring puckering in Shigella flexneri 7a and 7b O-antigens as a case study
    (2024) Timol, Zaheer; Kuttel, Michelle; Ravenscroft Neil
    This work forms part of a larger project investigating the conformation and dynamics of Shigella flexneri O-antigens. S. flexneri is a leading cause of diarrhoeal related diseases, especially in Sub-Saharan Africa and Southeast Asia. There are over 30 S. flexneri serotypes and, with the exception of serotype 6, all share a common O-antigen backbone (serotype Y), with variations in glucosylation, phosphorylation, and O-acetylation. O-antigens are carbohydrate polymers on the outer membrane of gram-negative bacteria. In S. flexneri, O-antigens are a primary antigenic component and are a target for conjugate vaccines currently in development. Analysis of O-antigen conformation for different S. flexneri serotypes may inform the vaccine development process. However, determining molecular conformation experimentally is challenging. Systematic molecular modelling protocols have proven useful in elucidating conformations of polysaccharide antigens, especially when experimental methods, such as nuclear magnetic resonance, are used for verification of modelling results. Here we use a combination of molecular modelling and 1H nuclear magnetic resonance spectroscopy experiments to probe the O-antigen conformations of S. flexneri 7a and 7b. Simulations of six repeating units of both O-antigens show that they are highly flexible, similar to the S. flexneri Y O-antigen. However, we found frequent puckering of the β-D-GlcpNAc ring away from the canonical 4C1 conformer, which has not been previously observed in studies of Shigella or other bacterial O-antigens. To provide further insight, molecular dynamics and metadynamics simulations of a range of 3,4-disubstituted β-D-GlcNAc trisaccharides with two carbohydrate force fields (CHARMM36 and GLYCAM06) were performed. The simulations reveal that 3,4-α-linked β-D-GlcpNAc puckers to a similar extent as the S. flexneri 7a and 7b O-antigens. Moreover, for both force fields the range of β-D-GlcNAc puckering is dependant on the anomeric configuration of both the 3- and 4- linkage, with non-4C1 conformations dominant in 3,4-α-linked β-D-GlcpNAc trisaccharides. 1H nuclear Overhauser effect spectroscopy experiments were used to calculate 1H-1H distances in S. flexneri 7a and 7b O-antigens. These experimentally derived distances match those calculated from the repeating unit simulations when a mix of 4C1 and boat/skew states of β-DGlcpNAc are considered at a ratio of 85:15. The results also suggest that puckering of β-DGlcpNAc does occur in S. flexneri 7a and 7b O-antigens but that it may be over represented in the simulations. Two general observations can be drawn from this study: (1) the 3,4-disubstitution of β-DGlcpNAc with two bulky substituents leads to the ring puckering out of the 4C1 conformer; and (2) the conformation and dynamic behaviour of β-D-GlcpNAc puckering is not accurately modelled by current methods, identifying the need for further enhancements to existing carbohydrate force fields.